Lai Dongmei, Cheng Weiwei, Liu Te, Jiang Lizheng, Liu Tianjin, Huang Qin, Guo Lihe
The International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, People's Republic of China.
Cell Reprogram. 2010 Jun;12(3):305-14. doi: 10.1089/cell.2009.0106.
Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by coculture with mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of animal sera such as fetal bovine serum (FBS). Here, we use primary human amnion epithelial cells (hAECs) as feeder cells and human umbilical cord blood serum (CBS) as a replacement for FBS to support undifferentiated growth of hESCs. The 5 approximately 10-fold higher expression levels of ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT were found in hESCs grown on hAECs compared with that on MEFs as measured by quantitative real-time polymerase chain reaction (PCR). By immunofluorescence, the expresisons of Oct-4 and Nanog is also higher in cells grown on hAECs than those on MEFs. Importantly, the ES cells grown on hAECs exhibit normal karyotypes on passage 25, thus ruling out the possibility of transformation. Using flow cytometry analysis, we show that both the ES cells grown on hAECs and MEFs have the same cell cycle distribution pattern. Further, hESCs cultured on hAECs for at least 20 passages could differentiate into three germ layers via teratoma formation. In addition, chromatin immunoprecipitation assay revealed that histone H3 is highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus and the Oct-4 locus in hESCs grown on hAECs. Conversely, hESCs grown on MEFs show histone deacetylation and H3-K4 demethylation. Taken together, these results suggest that hAECs supplemented with 10% CBS are suitable for hESC culture, and that this method may prove to be valuable for use in future regenerative therapies.
人胚胎干细胞（hESCs）通常通过与经丝裂霉素C处理的小鼠胚胎成纤维细胞（MEFs）共培养作为饲养层细胞，并在动物血清如胎牛血清（FBS）存在的情况下维持未分化状态。在此，我们使用原代人羊膜上皮细胞（hAECs）作为饲养层细胞，并用人脐带血血清（CBS）替代FBS来支持hESCs的未分化生长。通过定量实时聚合酶链反应（PCR）测量发现，与在MEFs上生长的hESCs相比，在hAECs上生长的hESCs中包括FGF、Oct-4、Nanog、Sox-2、Rex和TERT在内的ES细胞标志物的表达水平高出约5至10倍。通过免疫荧光检测，在hAECs上生长的细胞中Oct-4和Nanog的表达也高于在MEFs上生长的细胞。重要的是，在hAECs上生长的ES细胞在传代25次时表现出正常的核型，从而排除了转化的可能性。使用流式细胞术分析，我们表明在hAECs和MEFs上生长的ES细胞具有相同的细胞周期分布模式。此外，在hAECs上培养至少20代的hESCs可通过畸胎瘤形成分化为三个胚层。此外，染色质免疫沉淀分析表明，在hAECs上生长的hESCs中，组蛋白H3高度乙酰化，并且在Nanog基因座和Oct-4基因座处H3赖氨酸（K）4发生高甲基化。相反，在MEFs上生长的hESCs表现出组蛋白去乙酰化和H3-K4去甲基化。综上所述，这些结果表明补充有10%CBS的hAECs适合用于hESC培养，并且这种方法可能被证明对未来的再生治疗有价值。
